Surface plasmon resonance (SPR), using BIAcore2000 instruments, is utilized by our laboratory for studying protein-ligand interactions. For many years our laboratory has studied the interaction between antigens and antibodies and we have developed a novel experimental design using the surface plasmon resonance to resolve time-dependent kinetic behavior which is consistent with the formation of a complex, first as an encounter complex and changing into a more stable docked complex, evidencing at least two-states. This is the same model which recently has been proposed for time-dependent binding of the 90kDa heat shock protein (Hsp90) to inhibitors of the genaldenamycin (GA) family. Our previous experience with the antibody-antigen system therefore informs the study of new, uncharacterized systems such as Hsp90-GA interaction. The unique protocols we have developed in the long-term project on antibody-protein interactions will be the basis of several new initiatives addressing targeted Receptor-Ligand interactions important to cancer biology. The development of reliable methodology to time-dependent residency times is important to understanding pharmacological profiles of drugs many drugs. We have begun experiments to examine the time-dependent complex formation using SPR, and to develop general methodology for identifying time-dependent binding in other drug-target interactions. We have recently shown, using our model antibody system, that a mutant antigen with apparently lower binding affinity is actually a better inhibitor in SPR of antibody binding than the original antigen when examined in a long term competitive binding assay: binding inhibition correlates with length of preincubation time with the inhibitor. We have developed unique protocols for measuring binding kinetics and thermodynamics which show that the underlying mechanism is the slow, time-dependent formation of a very tight complex by the mutant inhibitor. This is the first reported demonstration of time-dependent inhibition efficicacy. We are planning experiments to also demonstrate this by isothermal titration calorimetry (ITC), which will require the development of additional novel ITC protocols. This experimental design can now be applied to drug-target interactions such as the Hsp90-GA interaction. The time-dependence of binding leading to long residence time of such inhibitors is likely to significantly impact their pharmalogical profile in patients, such as pharmacological effect and target selectivity. Thus development of reliable methodology to study this characteristic is likely of high impact In addition, we have recently found that uncomplexed single chain Fv fragments (scFv of anti-lysozyme antibodies undergo a concentration- and time-dependent, temperature-modulated change in affinity which we hypothesize represents a conformational transition. It was initially observed in scFv which had 6-19F-tryptophan incorporated into the 6 tryptophans of the binding site, and we developed a novel SPR protocol to study the temperature, time, and concentration dependence of the change. We then demonstrated that this occurs in unsubstituted scFv, and may be evidence of a conformational isomerism. Alternatively, it could be evidence of a concentration-dependent transient dimerism, but dynamic light scattering experiments indicate the protein is in a monomeric state, and the kinetics are not consistent with dimer binding. We expect by the end of this month to resolve this question using ultracentrifugation. These results are of clinical significance, because it may be a general phenomena of scFv, rather than specific to the antibody we are studying, and scFv reagents are used clinically for both diagnostic and therapeutic purposes. Thus, it is necessary to understand the effects of storing a scFv reagent at high concentrations and then subsequent dilution upon administration upon subsequent binding behavior when administered to a patient. Future Plans I expect to bring this research program to closure by September 30, 2009, which is the close of the FY09 budget period. This will entail completing experiments in progress, including the recent initiatives outlined above, and submitting results for publication.